Exploring the efficacy and molecular mechanism of Danhong injection comprehensively in the treatment of idiopathic pulmonary fibrosis by combining meta-analysis, network pharmacology, and molecular docking methods

Background: Danhong injection, a compound injection of Chinese herbal medicine, has been widely used in idiopathic pulmonary fibrosis (IPF) at present as an adjuvant treatment. However, the clinical efficacy and molecular mechanism of IPF are still unclear. This study will evaluate and explore the clinical efficacy and molecular mechanism of Danhong injection in the treatment of IPF. Methods: In meta-analysis, the computer was used to search 8 databases (PubMed, EMbase, CENTRAL, MEDLINE, CBM, CNKI, WanFang, and VIP) to collect the RCTs, and RevMan 5.3 and Stata 14.0 were used for statistical analysis. It has been registered on PROSPERO: CRD42020221096. In network pharmacology, the main chemical components and targets of the chemical components of Danhong injection were obtained in TCMSP and Swiss Target Prediction databases. The main targets of IPF were obtained through Gencards, Disgenet, OMIM, TTD, and DRUGBANK databases. The String platform was used to construct PPI networks. Cytoscape 3.8.2 was used to construct the “Danhong components – IPF targets-pathways” network. The molecular docking verification was conducted by Auto Dock. Results: Twelve RCTs were finally included with a total of 896 patients. The meta-analysis showed that Danhong injection could improve the clinical efficiency ([OR] = 0.25, 95% CI [0.15, 0.41]), lung function, arterial blood gas analysis, inflammatory cytokines, and serum cytokines associated with pulmonary fibrosis of IPF patients, respectively (P < .05). The core active components of Danhong injection on IPF were Luteolin, Quercetin, and Kaempferol, and the core targets were PTGS2, AR, ESR1, PPARG, and RELA. Danhong injection mainly improved IPF through PD-L1 expression and PD-1 checkpoint path in cancer, pathways in cancer, PI3K-Akt signaling pathway, etc. Conclusion: These results provided scientific basis for the clinical use of Danhong injection for the treatment of IPF, and provided a new direction to explore the potential mechanism of action of Danhong injection.


Introduction
Idiopathic pulmonary fibrosis (IPF) is an unexplained fibrotic interstitial pneumonia in which patients typically die within 2.5 to 5 years of definitive diagnosis. [1]It is characterized by the accumulation of proliferative fibroblasts and myofibroblasts, and a large amount of matrix in lung interstitium as the focus, and abnormal expression of the main components of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM: Collagen-I and Collagen-III). [2]IPF is characterized by high morbidity and mortality and has become a serious health problem. [3]The only approved drugs currently for the treatment of IPF are Pirfenidone and Nintedanib, however, these drugs can only delay the progression of IPF and do not improve the lesion. [1,4]Therefore, it is necessary to develop other new drugs or alternative drugs to treat IPF.
IPF belongs to the category of "Pulmonary flaccidity" in traditional Chinese medicine (TCM). [5]From the perspective of the pathogenesis of IPF, it is "blood stasis" essentially: blood stasis will lead to mutual resistance of sputum and stasis and eventually form IPF. Thus, blood stasis can be considered to be an important therapeutic target throughout the whole process of IPF. [5]Danhong injection contains 2 TCMs: Salvia miltiorrhiza Bunge (the dried root and rhizome of S miltiorrhiza) and Carthami Flos (the dried flower of Carthamus tinctorius L.).All the above plant names have been checked with http://mpns.kew.org.S miltiorrhiza Bunge, named Danshen in Chinese, has the functions of dredging meridians and relieving pain; Carthami Flos, named Honghua in Chinese, also has the functions of unblocking meridians and relieving pain, [6] and the combination of the 2 can complement each other, enhance the role of facilitating blood circulation and decreasing blood stasis.At present, as an adjuvant treatment to conventional treatment, Danhong injection has been widely used in cardiovascular disease, [7] cerebrovascular disease, [8] respiratory system disease, [9] and kidney disease [10] in China, and its curative effect is quite significant.[13] However, the clinical efficacy and molecular mechanism of its treatment of IPF are unclear.
This study combined meta-analysis, network pharmacology, and molecular docking to comprehensively evaluate and explore the clinical efficacy and molecular mechanism of Danhong injection in the treatment of IPF and provide strong evidence for it as a new alternative drug for IPF treatment.

Meta-analysis
This study has been registered on PROSPERO: CRD42020221096.

Inclusion criteria.
All included studies were randomized controlled trials (RCTs) of Danhong injection combining with conventional treatment for IPF, which must meet international [1] or Chinese [14] authoritative standards and be in English or Chinese.All patients were not restricted by gender, age, ethnicity, and nationality.Experimental group: combined use of Danhong injection on the basis of conventional treatment or control group.Control group: on the basis of conventional treatment, hormones, N-acetylcysteine (NAC), and other drugs were used.There were no restrictions on the dose, method of administration, and duration of the drugs in both groups.Conventional treatment included antibiotics and oxygen therapy, as well as other treatments.

Exclusion criteria.
Studies in which patients with other systemic serious diseases were excluded; non-RCTs, case reports, reviews, expert experience, and experimental studies were excluded; the results of the same study that has been published multiple times, only one of the most complete studies were included.
2. 1.4.Literature search strategy.Eight databases were searched by using theme words and keywords: PubMed, EMbase, CENTRAL, MEDLINE, CBM, CNKI, WanFang, and VIP Chinese Science, and the search date was set to November 2, 2022.Relevant literature in Google Scholar is manually retrieved as a supplement at the same time.Search terms: "IPF", or "Pulmonary fibrosis" or "Interstitial lung disease" or "Pulmonary interstitial fibrosis" or "IPF" and "Danhong" or "Danhong injection" and "Randomized controlled trial" or "RCT."Table S1, Supplemental Digital Content, http://links.lww.com/MD/M461 shows the search strategies in PubMed.

Literature screening and data extraction.
The literatures were independently screened by 2 researchers (Wu and Li).Full-text readings were conducted for potentially eligible entries.Literatures were handed over to a third party (Chen) to decide whether to really include the statistics when there were different opinions after reading the full text.For literatures with incomplete data, the original author was contacted by email for details.The data tables were designed by following the principle of "PICOST" (participants, interventions, comparisons, outcomes, study design, and timing).
2.1.6.Quality evaluation and bias risk assessment.The quality of the included studies and the risk of bias were assessed by using the modified Jadad Scale [16] and Cochrane 5.1.0.

Statistical analysis.
In this study, all statistical analysis was performed in Revman 5.3 and Stata 14.0.The dichotomous variables were statistically analyzed by using odds ratio (OR) and 95% confidence interval (95% CI), and the continuous variables were statistically analyzed by using mean difference (MD) and 95% CI.When there was heterogeneity between the data (P < .1,I 2 > 50%), the random effect model was used for analysis, on the contrary, the fixed-effect model was used.The Begg test and the Egger test were used to analyze publication bias.When there is obvious heterogeneity between the data, sensitivity analysis was conducted.www.md-journal.com2.2.Network pharmacology and molecular docking 2.2.1.Screening of targets of Danhong injection.In the traditional Chinese medicine system pharmacology (TCMSP) platform, [17] the chemical components of S miltiorrhiza Bunge (Danshen) and Carthami Flos (Honghua) in Danhong injection were searched.The active ingredients were initially screened according to the ADME attribute values (oral bioavailability (OB) ≥ 30% and Drug-likeness (DL) ≥ 0.18), and the targets of these active ingredients were obtained in TCMSP.The 2D molecular structures of these active ingredients were entered into Swiss Target Prediction database [18] and were screened at the same time to obtain the predicted targets of these components, and when the results display a higher probability value, it means a higher probability.

Screening of targets of IPF.
In the GeneCards database, disgenet database, OMIM database, TTD database, DRUGBANK database, "IPF" was entered and screened for potential targets for the treatment of IPF.According to the previous literature, [19] higher score value in the Genecards and disgenet databases indicated that these targets were closely associated with the disease.Therefore, if there are too many targets, the targets whose Score was greater than the median were set as the potential targets for IPF.Finally, After merging the targets of 5 disease databases and removing duplicate values, potential targets for IPF were obtained.

Establish PPI network for main targets of Danhong injection-IPF.
The jvenn online platform [20] was used to intersect the predicted targets of Danhong injection and the potential targets of IPF, and the Venny diagram was drawn at the same time, and then the intersection targets were submitted to the STRING11.5database to establish the protein-protein interaction (PPI) network model [21] : the biological species was set to "Homo sapiens," the minimum interaction threshold was set to "medium confidence > 0.4," and the rest of the settings were the default settings.The intersection targets greater than the median degree value were selected and determined to be the main targets of Danhong injection acting on IPF according to the size of the degree value, and they were further submitted to the STRING11.5database to obtain the PPI network of the main targets (the minimum interaction threshold was set to "highest confidence > 0.9"), and the PPI network was further analyzed through the Metascape platform to obtain the potential protein functional modules, and the function was described by analyzing the biological processes in which it participates.

Enrichment analysis of targets functions and pathways of Danhong injection-IPF.
With the aim of enrichment analysis of the main biological processes and signaling pathways of these targets, the main targets of Danhong injection acting on IPF were entered into the Metascape platform, [22] and P < .01 was set at the same time, and finally the data results were saved and visualized by using the ehbio online platform.The enrichment analysis consisted of 4 parts: BP for biological processes of Danhong injection on IPF, MF for molecular functions, CC for cellular components, and KEGG for signaling pathways and the main targets contained in these pathways. [23]2.5.Construction of Danhong injection-IPF targetssignal pathway network diagram.CytoScape 3.8.2 was used to construct the network diagram of the main targetspathway of Danhong injection-IPF, and the network topology parameters of the active ingredients and targets, including Degree, Betweenness, and Closeness, were analyzed by using the built-in tools of CytoScape3.8.2, and the core active ingredients, core targets and core pathways of Danhong injection in the treatment of IPF were judged according to the network topology parameters.
2.2.6.Molecular docking verification.The top 5 targets in the network diagram of the main targets of Danhong injection-IPF were molecularly docked with the core active ingredients of Danhong injection: first, the 3D structure PDB format files of 5 target proteins were downloaded from the RSCB PDB database, and second, the SDF format files of the 2D structure of the core active ingredients of Danhong injection were downloaded from the PubChem database and converted into mol2 format files by using Open Babel GUI software.Molecular docking was carried out in Auto Dock software: first, the target proteins were dehydrated and hydrogenated.Second, Auto Dock software was used to convert the format files of Danhong injection's core active ingredients and target proteins into PDBQT.Finally, run the Auto Dock program for molecular docking.If the binding energy is <0, it means that the ligands and the receptors can bind spontaneously.

Quality evaluation and bias risk assessment.
Data from all 12 RCTs were complete, and they all used randomization methods.These RCTs all described the comparability of baseline data between the experimental and control groups in detail, and they all described the therapeutic measures and efficacy outcomes in detail.However, they did not specify the method of allocation concealment or blinding.The evaluation results of the Jadad scale [16] are shown in Table S2, Supplemental Digital Content, http://links.lww.com/MD/M462: 8 studies were with 2 points (low-quality) and 4 studies were with 3 points (mediumquality). [24,25,29,32]The low and medium risks resulting from random sequences accounted for 32% and 68% of the selection bias of the original study, respectively (Figures S1 and S2, Supplemental Digital Content, http://links.lww.com/MD/M445,http://links.lww.com/MD/M446).Therefore, there are certain biases in the included studies, such as selection, implementation, and measurement bias.

Adverse reactions.
One study [24] reported gastrointestinal discomfort in 3 patients in the Danhong injection group, but this was mainly due to the use of erythromycin.There were no obvious adverse reactions in other patients.In addition, 2 studies [27,32] reported no adverse reactions and the other 9 studies did not describe adverse reactions.

Target acquisition of active components of Danhong injection.
Two hundred two chemical components of S miltiorrhiza Bunge (Danshen) and 189 chemical components of Carthami Flos (Honghua) were preliminarily extracted.After ADME screening, 65 active components of S miltiorrhiza Bunge (Danshen) and 22 active components of Carthami Flos (Honghua) were obtained, and a total of 84 active components of Danhong injection were obtained after combining them (Table S6, Supplemental Digital Content, http://links.lww.com/MD/M466).There were 349 targets of active components of S miltiorrhiza Bunge (Danshen) and 296 targets of active components of Carthami Flos (Honghua).After the combination, a total of 476 targets of Danhong injection were obtained by deleting the duplicate values.

Acquisition of IPF-related targets.
The targets obtained in 5 databases were combined, and the duplicate values were removed, and 1870 IPF-related targets were finally obtained.As shown in Table S7, Supplemental Digital Content, http://links.lww.com/MD/M467.

PPI network construction of Danhong injection-IPF targets.
The 476 targets of the screened Danhong injection were intersected with the 1870 disease targets of IPF, and 221 intersecting targets were obtained, as shown in Figure 4. Furthermore, 221 intersecting targets were submitted to the STRING11.5platform, and the degree values of these 221 intersecting targets were calculated according to the node degrees module in the platform, and 114 targets greater than the median (≧42) were set as the main targets of Danhong injection acting on IPF according to the median of the degree values.After obtaining the main targets, they were further submitted to the STRING11.5platform and the PPI network diagram of the main targets of Danhong injection acting on IPF was obtained (Fig. 5).The interaction relationship of these main targets was analyzed by molecular complex detection algorithm to obtain the modules in PPI networks [37] in Metascape (Fig. 6), and the biological processes that retain the 3 best scores in the Modules according to the P values were described functionally (Table 3).

Enrichment analysis of target functions and pathways.
The Metascape was used to perform functional and pathway analysis of 114 main targets of Danhong injection acting on IPF, and the results were visualized with the help of the ehbio online platform.The results showed that the main biological processes of Danhong injection on IPF included response to hormone, positive regulation of cell migration, cellular response to lipid, response to inorganic substance, and response to growth factor, etc (Fig. 7A).The main molecular Chen [25]  Wang [30]  Wang [31]  functions included kinase binding, transcription factor binding, cytokine receptor binding, protein domain specific binding, and protein kinase activity, etc (Fig. 7B).The main cellular components included membrane raft, transcription regulator complex, side of membrane, platelet alpha granule, perinuclear region of cytoplasm, etc (Fig. 7c).The main signaling pathways included Pathways in cancer (PC), lipid and atherosclerosis (LA), AGE-RAGE signaling pathway in diabetic complications (ARPDC), proteoglycans in cancer (PRC), PI3K-Akt signaling pathway (PASP), etc (Fig. 7D and E), and more detailed enrichment results are shown in    ESR1, PPARG, and RELA).The results showed that the binding energies of the core active ingredients to the targets were <0 (<0 kJ/mol −1 ), which indicated that the active ingredients in Danhong injection had good binding activity to the core targets of IPF.As shown in Table 7 and Figure 9.

Discussion
In this study, the clinical efficacy and safety of Danhong injection in the treatment of IPF were evaluated by meta-analysis, and the targets and molecular mechanism of Danhong injection in the treatment of IPF were further explored by combining network pharmacology and molecular docking methods.The obtained results were satisfying.In meta-analysis, the results confirmed that Danhong injection, as an adjuvant treatment, performed better in improving clinical efficacy, lung function (FEV 1 /FVC%, FVC(L)), analysis of the arterial blood gas (DLCO, PaO 2 ), inflammatory cytokines (TNF-α, TGF-β 1 ), and serum cytokines associated with pulmonary fibrosis (HA, LN, PCIII, ColIII, BUN), which is suggesting that Danhong injection has certain advantages and curative effects as a alternative drug for the treatment of IPF.Regarding the safety of Danhong injection, there was only 1 description of adverse reactions in all the literatures, but the main cause was the side effects of the combination of medications, and the incidence of adverse reactions in patients was negligible.In addition, 2 other studies showed no adverse reactions.These results show that Danhong injection has a good safety profile.Some researchers used systems biology methods to simulate the mechanism of the compatibility of S miltiorrhiza Bunge and Carthami Flos (The 2 main Chinese medicines contained in Danhong injection) and found that they may play synergistic roles in the treatment of diseases mainly in biological pathways such as NF-κB cascade reaction, autophagy, proliferation, and migration. [38]Some studies also showed that Danhong injection exerted a multi-target antiinflammatory effect by inhibiting the expression of tumor necrosis factor-α (TNF-α) and other cytokines. [39]owever, the targets and molecular mechanism of Danhong injection in the treatment of IPF are still unclear.Therefore, we further explored the targets and molecular mechanism of Danhong injection in the treatment of IPF.In the network pharmacology, the targets of the main components of Danhong  injection and the targets of IPF were screened and it was found that there were 221 related intersection target proteins between Danhong injection and IPF.Subsequently, PPI interaction analysis and the construction of the main components of Danhong injection-IPF main targets-pathways network were carried out, and the core components, core targets, and main signaling pathways of Danhong injection acting on IPF were obtained, and the molecular docking verification of the core components and core targets of Danhong injection acting on IPF was carried out.
Network pharmacological results showed that the core components of Danhong injection acting on IPF were mainly concentrated in Luteolin, Quercetin, and Kaempferol.Luteolin was a natural flavonoid that was widely found in many plants. [40,41]uteolin has a variety of biological effects, such as antiinflammatory, antioxidant, and anticancer. [42]Luteolin was found to improve collagen deposition effectively, transforming growth factor-β1 (TGF-β1) expression and pulmonary fibrosis in the lung tissues of mice induced by bleomycin (BLM), and similar results were found in vitro studies.The mechanism of these results was related to attenuation of TGF-β1-induced phosphorylation of Smad549.It is been shown that Luteolin can exert anti-pulmonary fibrosis effects by inhibiting lung inflammation and inhibiting myofibroblast differentiation and epithelial to mesenchymal transformation. [43]In terms of anticancer, studies have shown that Luteolin hinder cancer progression through a variety of mechanisms, including inhibition of protein kinase, regulation of cell cycle, induction of apoptosis, induction of DNA damage, and reduction of transcription factors. [44,45]In addition, in the model of lipopolysaccharide (LPS)-induced acute lung injury in mice, Luteolin suppressed the inflammatory response in lung tissue in a concentrationdependent manner and reduced the levels of TNF-α, IL-6, iNOS, and cyclooxygenase-2 (COX-2).The mechanism was related to the inhibition of the activation of NF-κB and its upstream molecular factor Akt. [46] Luteolin can also prevent liver fibrosis by acting on the toll-like receptor (TLR) signaling pathway to reduce extracellular matrix accumulation and cathepsin gene expression while enhancing the liver's antioxidant system. [47]s a natural polyhydroxyflavonoid, Quercetin was widely present in nature.It has a variety of physiological activities such as oxidative stress, reducing inflammatory response, antitumor, cardiovascular protection, antihypertensive, antithrombotic, anti-adhesion, anti-atherosclerotic effects, etc. [48][49][50] It also has intracellular antioxidant activity that inhibits the production of hydrogen peroxide-induced intracellular ROS in mouse fibroblasts, thereby mitigating diseases caused by oxidative stress. [51]n the BLM-induced pulmonary fibrosis model of rats, it was found that the antioxidant capacity of rat lung tissue was significantly reduced, and the histopathological structure of lung was significantly improved after the intervention with Quercetin. [52]n addition, Quercetin had anti-inflammatory effects in rats and patients with IPF, as evidenced by its ability to reduce damage caused by inflammatory cell infiltration and reduce the amount of inflammatory factors TNF-α and IL-8. [53,54]It also inhibits the progression of PF by inhibiting the proliferation of mouse alveolar type II epithelial cells and NIH3T3 cells induced by TGF-β1 signaling, thereby reducing collagen and hyaluronic acid secretion [55] ; it can improve PF by inhibiting the expression of TGF-β1-induced α-SMA, fibronectin, and collagen by affecting SphK1 or S1P3/SphK signaling pathways [56,57] ; And it can upregulate the expression of FasL receptor and reverse the resistance of fibrotic cells to apoptosis, thereby alleviating BLM-induced PF in mice [58] ; it also promotes the secretion of MMPs by human embryonic lung fibroblasts, restoring the balance of MMP-1/ TIMP-1 to reduce lung fiber maintenance. [59]Kaempferol was a natural flavonoid found widely in many fruits and vegetables [60] with anti-inflammatory, antioxidant, and antibacterial effects. [61]aempferol has been found to alleviate fibrotic airway remodeling by inactivating PAR1 in epithelial cells, [62] and to protect  mice from acute lung injury by regulating TRAF6 ubiquitination [63] and by inhibiting the expression of oxidative stress proteins iNOS (NOS2) and ICAM-1. [64]In addition, Kaempferol has shown significant antifibrotic effects in cardiac remodeling [61] and it can also inhibit the progression of silica-induced PF. [65] Therefore, the core active ingredients of Danhong injection (Luteolin, Quercetin, and Kaempferol) mainly improve IPF by anti-inflammatory, oxidative stress, induction of apoptosis, inhibition of cell proliferation, regulation of immunity, induction of DNA damage, reduction of transcription factors, etc. Network pharmacological results showed that the main targets of Danhong injection on IPF were PTGS2, AR, ESR1, PPARG, RELA, etc. Prostaglandin-endoperoxide synthase 2 (PTGS2) is the gene encoding cyclooxygenase-2 (COX-2), which is an inducible enzyme that can participate in various pathological processes such as inflammation, cell proliferation, and apoptosis in various cells. [66]It is been reported that COX-2 was highly expressed in bleomycin-induced pulmonary fibrosis tissues. [67]Androgens can increase the androgen receptor (AR) protein levels in lung cells, and AR was mainly expressed in alveolar epithelial cells (AECs) II and bronchial epithelial cells of mouse lungs.In lung cancer studies, TMPRSS2 has been found to be one of the genes upregulated by androgen exposure in A549 cells and is a direct target of AR. [68] Estrogen (E2) (17β-estradiol) can activate several receptors, including the nuclear transcription factor estrogen receptor α (ESR1) and estrogen receptor β (ESR2).ESR1 expression has been found to be higher than ESR2 in human lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), suggesting that ESR1 may play a major role in inducing lung cancer (LC) cells. [69]However, in another study, exposure to TGF-β1 in bronchial epithelial cells resulted in dose-dependent and significantly reduced ESR1 and ESR2 mRNA and protein expression. [70]Therefore, whether ESR1 has a protective or inducing effect on pulmonary fibrosis is controversial.PPARG encoded peroxisome proliferator-activated receptor γ (PPARγ), and the antifibrotic effect of PPARγ has been found to be manifested by inhibiting TGF-β expression and blocking downstream signal transduction after ligand activation. [71]Furthermore, Metformin induced adipose differentiation in myofibroblasts by upregulating BMP2 and activating PPARγ. [72]These results indicate the progression of negative regulatory fibrosis by activation of PPARγ and suggest that this receptor may be a key target for the treatment of IPF.The NF-κB signaling pathway is involved in inflammatory response, cell proliferation, cell differentiation, cell adhesion and growth signaling, apoptosis defense, and host immune response. [73]There are many basic transcription factors in the NF-κB signaling pathway, among which the gene encoding transcription factor p65 is RELA. [74]Studies have shown that the inhibition of autophagy in alveolar epithelial cells of IPF patients is accompanied by p65/RELA-mediated activation of SNAI2, a process that not only controls epithelial-mesenchymal transition (EMT), but also regulates the production of locally acting profibrotic mediators. [75]In addition, NF-κB/RelA was found to be the major mediator of TGF-β-induced EMT in vitro models of telomerase immortalized epithelial cells [76] : TGF-β activated NF-κB/RelA and induced its nuclear translocation, and NF-κB/RelA can directly upregulate multiple core EMT regulators in the nucleus. [77]Therefore, the main functions of PTGS2, AR, ESR1, PPARG, and RELA in the formation of IPF can be summarized as: pro-inflammatory response, regulation of cell adhesion and growth signals, control of cell proliferation, differentiation and apoptosis, regulation of oxidative stress response, and regulation of host immune response.These functions are consistent with the effect of the core active ingredients (Luteolin, Quercetin, and Kaempferol) of the aforementioned Danhong injection in improving IPF, and the results of molecular docking verification also show that Luteolin, Quercetin, and Kaempferol have strong binding activity to these targets.Therefore, it can be considered that Danhong injection mainly acts on PTGS2, AR, ESR1, PPARG, RELA, and other targets to treat IPF.In network pharmacology, KEGG pathway analysis found that Danhong injection was mainly through PD-L1 expression and PD-1 checkpoint pathway in cancer (PPCPC), LA, proteoglycans in cancer (PRC), PC, PASP, etc to treat IPF.Studies showed that although the cell origin and pathological phenotype were different, the pathogenesis of IPF and LC was highly similar, they both have chronic damage to AECs accompanied by abnormal tissue repair and alveolar structure damage. [78,79]herefore, it can be considered that the mechanism of IPF and cancer production is similar, and related cytokines can act on PC and PRC to induce IPF production.Programmed death receptor 1 (PD-1) on T cells is involved in regulating T cell function under normal physiological conditions. [80]After the activation of effector T cells, the programmed death ligand 1 (PD-L1) of PD-1 is upregulated in peripheral tissues. [80,81]Subsequent binding of PD-1 to PD-L1 results in inhibitory co-stimulation signaling of T cell receptors (TCRs), a process that is essential for maintaining immune tolerance and preventing autoimmune pathology. [82]Studies have found that PD-L1 expression is present in lung fibrosis-related tumor cells of 62% of patients and the PD-1/PD-L1 axis is induced.These results confirmed the potential role of PD-1/PD-L1 checkpoint in IPF. [83]Moreover, serum expression of soluble PD-L1 had increased threefold in patients with IPF. [84]In addition, other studies found that the expression of PD-L1 in peripheral blood of IPF patients did not increase, but the expression of PD-1 in T lymphocytes in peripheral blood and lung tissue of IPF patients increased significantly. [85]These studies suggest that PD-1/PD-L1 checkpoints are induced in IPF regardless of the actual cell type expressing PD-1 or PD-L1, therefore immune editing may affect the disease progression of IPF.Most vascular resistance in pulmonary fibrosis is due to persistent vasoconstriction, [86] which has some common ground with pulmonary hypertension (PAH).Many of the pathophysiological mechanisms found in PAH overlap with those associated with atherosclerosis, including vascular smooth muscle and endothelial cell dysfunction. [87]Furthermore, prostacyclin (PGI 2) has been found to play a central role in the treatment of PAH through its powerful vasodilating, antithrombotic, and antiproliferative effects. [88]Therefore, the pathological process of IPF is also related to the mechanism of atherosclerosis.The PI3K/ Akt/mTOR signaling pathway is one of the important signal transduction pathways in cells, which plays an important role in regulating physiological functions such as cell growth, proliferation, motility, cell survival, autophagy, protein synthesis, and  transcription. [89]In PI3K/Akt/mTOR, phosphoesterinositol-3kinase (PI3K) is a signal transductase that is the initiation factor of the PI3K/Akt/mTOR pathway. [90]Moreover, protein kinase B (PKB: alias Akt) is located in downstream of PI3K, and it can activate downstream mTOR and others to participate in cell differentiation and proliferation [91,92] when it is phosphorylated.
Studies has shown that it plays a key regulatory role in the formation of IPF: PI3K/Akt/mTOR is highly expressed in AECs II in the IPF rat model, thereby inhibiting autophagy, promoting collagen synthesis, and IPF. [93]Therefore, these signaling pathways mainly play the role of pro-inflammatory response, regulation of oxidative stress response, regulation of immune response, control of apoptosis, cell proliferation, and regulation of vascular endothelial cell function in the formation of IPF.Combined with the above results, it can be considered that Danhong injection is mainly expressed through PPCPC, LA, and PRC, PC, PASP act on PTGS2, AR, ESR1, PPARG, RELA, and other targets to treat IPF.
Limitations of the study: meta-analysis: all 12 literatures only mentioned "random," but did not indicate a specific method of randomization.In addition, no literature reported on allocation concealment and blinding.Therefore, the literature included in this meta-analysis may be biased; these studies were unicentre and had small sample sizes.In addition, they have some clinical heterogeneity, such as a different course of treatment and a different course of disease.These reasons may reduce the reliability of this meta-analysis; all the studies had no report of relevant death and endpoint indicators, and no description of the patient's follow-up, so it is nearly impossible to reach the conclusion that whether Danhong injection can better reduce the patient's mortality and other endpoint events or not.Network pharmacology: based on the results of bioinformatics and massive data calculation, this study preliminarily elucidated the mechanism of the action of Danhong injection in the treatment of IPF from the perspective of network pharmacology.However, since most of the databases, such as GeneCards, derive their data on disease-associated genes from results reported in a portion of the previous literature, their respective data may not be particularly complete.Therefore, there may be inconsistencies between the results of these databases and those reported in the few available literature.These results should be subsequently validated by in vivo or in vitro experiments to further clarify the main targets and molecular mechanisms of Danhong injection in regulating IPF.

Figure 2 .
Figure 2. The forest plot of clinical efficacy.

Figure 3 .
Figure 3.The funnel plot of clinical efficacy.

Table 3 Function
description of PPI network of Danhong injection -IPF targets.cell motility −50.7 www.md-journal.com
PASP to play the role of anti-inflammatory, anti-oxidative stress, inducing apoptosis, inhibiting cell proliferation, regulating immunity, inducing DNA damage and reducing transcription factors to reduce fibronectin and collagen, inhibit extracellular matrix deposition and airway remodeling to improve IPF.This conclusion provides a scientific basis for the clinical use of Danhong injection for the treatment of IPF and provides a new direction to explore the potential mechanism of action of Danhong injection.

Table 1
Basic features of the included studies.
Twenty major pathways and related targets were introduced into CytoScape3.8.2 to construct the network diagram of the main component of Danhong injection-IPF main target-pathway, as shown in Figure8.The network topology parameters of Danhong injection acting on IPF were analyzed by the built-in Network Analyzer of CytoScape3.8.2, and the core components, core targets, and core signaling pathways of Danhong injection acting on IPF were obtained.The results showed that Luteolin had a Degree of 74, Betweenness Centrality of 0.0338, and Closeness Centrality of 0.4592, therefore Luteolin was predicted to be the core component of Danhong injection to improve IPF, followed by Quercetin and Kaempferol, as shown in Table4.The results showed that PTGS2 had a Degree of 75, Betweenness Centrality of 0.29, and Closeness Centrality of 0.5714 in the network, therefore it is predicted that PTGS2 was the core target of Danhong injection on IPF.

Table 2
The results of meta-analysis of secondary outcomes.

Table 5
Characteristic parameters of target nodes of main active components of Danhong injection (Top 14).

Table 6
Characteristic parameters of target nodes of main active components of Danhong injection (Top 10).

Table 7
Binding energies of molecular docking of core compounds in Danhong injection.